Plasmid plus maxiprep or midiprep kits

Editor plasmids used for mammalian cell transfection were generated using Gibson assembly or USER assembly using pCMV-PE2 (Addgene, 132775) and pCMV-PEmax (Addgene, 174820) plasmids. pegRNA and epegRNA constructs were cloned by Golden Gate assembly using custom pU6-pegRNA-GG-acceptor (Addgene,132777) and pU6-tevopreQ1-GG-acceptor (Addgene, 174038) plasmids. All nicking sgRNA plasmids were generated by KLD assembly or Golden Gate assembly using pFYF1320 (Addgene, 47511) as a template plasmid. rAAV vector plasmids were cloned by restriction digestion of v5 AAV CBE (Addgene, 137176) or v5 AAV ABE (Addgene, 137177) followed by Gibson assembly with eBlocks or polymerase chain reaction (PCR) amplicons. All plasmids used for mammalian tissue culture were purified from MACH1, DH5alpha or NEBstable Escherichia coli using Plasmid Plus Maxiprep or Midiprep kits (Qiagen), ZymoPURE II Midiprep Kit (Zymo Research) or PureYield Plasmid Miniprep kit (Promega).

Expression vectors for tissue culture were cloned using KLD, Gibson or USER assembly. sgRNA expression plasmids were cloned via KLD or Goldengate assembly to install protospacers as indicated in Supplementary Table 1. Plasmids were constructed via USER assembly or Gibson assembly of PCR-amplified fragments. Plasmids encoding recombinant AAV (rAAV) genomes were cloned by Gibson assembly of plasmid restriction fragments and PCR amplicons with Gibson-compatible overhangs. All plasmids for mammalian tissue culture experiments were purified using Plasmid Plus Maxiprep or Midiprep kits (Qiagen), ZymoPURE II Midiprep kit (Zymo Research) or PureYield Plasmid Miniprep kits (Promega). Key plasmids developed in this study will be available through Addgene.